Myosin V is a two-headed unconventional myosin that has an extended neck due to the presence of six light chain binding IQ-repeats. Double- headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V, each tagged with the FLAG-epitope on their carboxyl-terminal end, were expressed in Sf9 cells. The cells were coinfected with three baculoviruses containing the appropriate heavy chain, calmodulin and essential light chain. The expressed proteins were purified using FLAG-affinity column chromatography. The myosin V fragments had a high Vmax and a low KATPase of the steady state actin- activated MgATPase. There was no activation of the MgATPase activity by calcium. The KATPase for the fragments were remarkably insensitive to ionic strength compared to fragments of rabbit skeletal muscle myosin. Similarly, myosin V translocated actin filaments over a wide range of ionic strengths in an in vitro motility assay and could do so at very low surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. In 200mM ATP, the sliding velocity was inhibited to 50% of its maximum at 25 micormolar ADP. Transient kinetic experiments revealed that ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 per sec at 20 C. The Vmax of the actin activated MgATPase activity at this temperature was 3.3 per sec, indicating that, while not totally rate limiting, ADP dissociation was close to the rate limiting step. The high affinity for actin and the slow rate of ADP release helps to assure a high duty cycle ratio and allows actin filaments to be moved by only a few myosin V molecules in vitro. Inside the cell, this means that few myosin V molecules would be required to translocate vesicles on actin filaments. In collaboration with Drs. Justin Molloy and Claudia Veigel of University of York, UK, we are performing single molecule mechanical studies. These data indicate that myosin V is able to move actin about 20 nm per step and that it has a very long attached lifetime. In addition, the step is composed of two substeps of dissimilar magnitude. - myosin V; in vitro motility; optical trapping;actin-activated MgATPase; stopped- flow fluorimetry